A closed compact structure of native Ca(2+)-calmodulin

Calmodulin has been a subject of intense scrutiny since its discovery because of its unusual properties in regulating the functions of about 100 diverse target enzymes and structural proteins. The original and to date only crystal conformation of native eukaryotic Ca(2+)-calmodulin (Ca(2+)-CaM) is a very extended molecule with two widely separated globular domains linked by an exposed long helix. Here we report the 1.7 A X-ray structure of a new native Ca(2+)-CaM that is in a compact ellipsoidal conformation and shows a sharp bend in the linker helix and a more contracted N-terminal domain. This conformation may offer advantages for recognition of kinase-type calmodulin targets or small organic molecule drugs.     

Crystal structure of kinesin regulated by Ca(2+)-calmodulin

Kinesins orchestrate cell division by controlling placement of chromosomes. Kinesins must be precisely regulated or else cell division fails. Calcium, a universal second messenger in eukaryotes, and calmodulin regulate some kinesins by causing the motor to dissociate from its biological track, the microtubule. Our focus was the mechanism of calcium regulation of kinesin at atomic resolution. Here we report the crystal structure of kinesin-like calmodulin-binding protein (KCBP) from potato, which was resolved to 2.3 A. The structure reveals three subdomains of the regulatory machinery located at the C terminus extension of the kinesin motor. Calmodulin that is activated by Ca2+ ions binds to an alpha-helix positioned on the microtubule-binding face of kinesin. A negatively charged segment following this helix competes with microtubules. A mimic of the conventional kinesin neck, connecting the calmodulin-binding helix to the KCBP motor core, links the regulatory machine to the kinesin catalytic cycle. Together with biochemical data, the crystal structure suggests that Ca(2+)-calmodulin inhibits the binding of KCBP to microtubules by blocking the microtubule-binding sites on KCBP.     

Ca(2+)-calmodulin regulates receptor-operated Ca(2+) entry activity of TRPC6 in HEK-293 cells

Mammalian homologues of the Drosophila transient receptor potential channel (TRPC) are involved in Ca(2+) entry following agonist stimulation of nonexcitable cells. Seven mammalian TRPCs have been cloned but their mechanisms of activation and/or regulation are still the subject of intense research efforts. It has already been shown that calmodulin (CaM) can regulate the activity of Drosophila TRP and TRPL and, more recently, CaM has been shown to interact with mammalian TRPCs. In this study, TRPC6 stably transfected into HEK-293 cells was used to investigate the possible influence of CaM on TRPC6-dependent Ca(2+) entry. Overexpression of TRPC6 in mammalian cells is known to enhance agonist-induced Ca(2+) entry, but not thapsigargin-induced Ca(2+) entry. Here, we show that CaM inhibitors (calmidazolium and trifluoperazine) abolish receptor-operated Ca(2+) entry (ROCE) without affecting thapsigargin-operated Ca(2+) entry and that the activity of CaM is dependent on complexation with Ca(2+). We also show that Ca(2+)-CaM binds to TRPC6 and that the binding can be abolished by CaM inhibitors. These results indicate that CaM is involved in the modulation of ROCE.     

Unusual Ca(2+)-calmodulin binding interactions of the microtubule-associated protein F-STOP

F-STOP is a microtubule-associated protein that stabilizes microtubules in a calmodulin (CaM)-dependent manner. All members of the stable tubule only polypeptide (STOP) family have a central domain that contains nearly identical multiple repeats, and a CaM binding motif is present in multiple copies within this domain. We present here an analysis of this CaM binding interaction and find that it is highly unusual in nature. For this work, we synthesized two model peptides of a single STOP central repeat motif and analyzed their binding to CaM by fluorescence, circular dichroism, infrared and NMR spectroscopy. Both peptides bind to CaM with an affinity of 4 microM, similar to that of the native protein. Results indicate that the peptides bind CaM in an atypical manner. Binding is highly dependent on the concentration of cations, indicating that it is to some extent electrostatic. Further, IR and CD analysis shows that, in contrast to typical CaM binding reactions, CaM does not change in helical structure on binding. NMR mapping confirms that CaM remains in extended conformation on binding a single STOP peptide. Binding of a single peptide to CaM occurs principally in the CaM C-terminal region, and the C-terminal domain of CaM effectively competes for STOP binding. Our results establish that CaM binds STOP in an unusual manner, involving mainly the C-terminus of CaM, thus leaving CaM potentially accessible for another binding partner at the N-terminus. This intriguing possibility could be of physiological importance in F-STOP mediated CaM regulation of microtubule dynamics or stability, specifically during mitosis where CaM and STOP colocalize.     

Ca(2+) dissociation from the C-terminal EF-hand pair in calmodulin: a steered molecular dynamics study

We used steered molecular dynamics (SMD) to simulate the process of Ca²⁺ dissociation from the EF-hand motifs of the C-terminal lobe of calmodulin. Based on an analysis of the pulling forces, the dissociation sequences and the structural changes, we show that the Ca²⁺-coordinating residues lose their binding to Ca²⁺ in a stepwise fashion. The two Ca²⁺ ions dissociate from the two EF-hands simultaneously, with two distinct groups among the five Ca²⁺-coordinating residues affecting the EF-hand conformational changes differently. These results provide new insights into the effects of Ca²⁺ on calmodulin conformation, from which a novel sequential mechanism of Ca²⁺-calmodulin dissociation is proposed.     

Differential codes for free Ca(2+)-calmodulin signals in nucleus and cytosol

BACKGROUND: Many targets of calcium signaling pathways are activated or inhibited by binding the Ca(2+)-liganded form of calmodulin (Ca(2+)-CaM). Here, we test the hypothesis that local Ca(2+)-CaM-regulated signaling processes can be selectively activated by local intracellular differences in free Ca(2+)-CaM concentration. RESULTS: Energy-transfer confocal microscopy of a fluorescent biosensor was used to measure the difference in the concentration of free Ca(2+)-CaM between nucleus and cytoplasm. Strikingly, short receptor-induced calcium spikes produced transient increases in free Ca(2+)-CaM concentration that were of markedly higher amplitude in the cytosol than in the nucleus. In contrast, prolonged increases in calcium led to equalization of the nuclear and cytosolic free Ca(2+)-CaM concentrations over a period of minutes. Photobleaching recovery and translocation measurements with fluorescently labeled CaM showed that equalization is likely to be the result of a diffusion-mediated net translocation of CaM into the nucleus. The driving force for equalization is a higher Ca(2+)-CaM-buffering capacity in the nucleus compared with the cytosol, as the direction of the free Ca(2+)-CaM concentration gradient and of CaM translocation could be reversed by expressing a Ca(2+)-CaM-binding protein at high concentration in the cytosol. CONCLUSIONS: Subcellular differences in the distribution of Ca(2+)-CaM-binding proteins can produce gradients of free Ca(2+)-CaM concentration that result in a net translocation of CaM. This provides a mechanism for dynamically regulating local free Ca(2+)-CaM concentrations, and thus the local activity of Ca(2+)-CaM targets. Free Ca(2+)-CaM signals in the nucleus remain low during brief or low-frequency calcium spikes, whereas high-frequency spikes or persistent increases in calcium cause translocation of CaM from the cytoplasm to the nucleus, resulting in similar concentrations of nuclear and cytosolic free Ca(2+)-CaM.     

Ca(2+) dynamics in the lumen of the endoplasmic reticulum in sensory neurons: direct visualization of Ca(2+)-induced Ca(2+) release triggered by physiological Ca(2+) entry

In cultured rat dorsal root ganglia neurons, we measured membrane currents, using the patch-clamp whole-cell technique, and the concentrations of free Ca(2+) in the cytosol ([Ca(2+)](i)) and in the lumen of the endoplasmic reticulum (ER) ([Ca(2+)](L)), using high- (Fluo-3) and low- (Mag-Fura-2) affinity Ca(2+)-sensitive fluorescent probes and video imaging. Resting [Ca(2+)](L) concentration varied between 60 and 270 microM. Activation of ryanodine receptors by caffeine triggered a rapid fall in [Ca(2+)](L) levels, which amounted to only 40--50% of the resting [Ca(2+)](L) value. Using electrophysiological depolarization, we directly demonstrate the process of Ca(2+)-induced Ca(2+) release triggered by Ca(2+) entry through voltage-gated Ca(2+) channels. The amplitude of Ca(2+) release from the ER lumen was linearly dependent on I(Ca).     

Ca(2+)-calmodulin regulated effectors of microtubule stability in neuronal tissues.

In general, microtubules are labile structures which depolymerize at low temperature and are sensitive to Ca2+. However, in brain tissue, axonal microtubules are disassembly-resistant and can exist without attachment to a microtubule organizing center. Stable microtubules cannot be purified by usual recycling procedures and this has made the elucidation of the molecular mechanisms involved in their stabilization difficult. This paper summarizes previous work in our laboratories, aimed at the identification of brain microtubule stabilizing proteins. We present assay methods which allow the detection of microtubule stability effectors in complex extracts and in chromatographic column fractions. Applied to brain crude extracts, they result in the isolation of Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated proteins. One, called STOP, appears to account for microtubule stabilization in neurons. A second protein with similar activity is myelin basic protein. Non-neuronal tissues also contain Ca(2+)-calmodulin-regulated effectors which appear to differ in structure from their neuronal counterparts. Thus, in all tissues examined, microtubule stability seems to be accounted for by unique Ca(2+)-calmodulin regulated proteins, showing tissue specificity.     

Ca(2+)-calmodulin regulated effectors of microtubule stability in bovine brain.

Stable microtubules (as defined by resistance to Ca2+, drug or cold temperature induced disassembly) form in abundance during tubulin assembly in brain crude extracts. We have previously shown that, in rat brain crude extracts, all microtubule stabilizing activity could be ascribed to a single Ca(2+)-calmodulin binding and Ca(2+)-calmodulin regulated protein, called "stable tubule only polypeptide", STOP145 [Pirollet, F., Rauch, C. T., Job, D., & Margolis, R. L. (1989) Biochemistry 28, 835-842]. We have now performed an exhaustive study of STOP-like effectors in bovine brain high-speed supernatants. All activity binds to cation exchangers and to Ca(2+)-calmodulin affinity columns. The activity can be resolved into two peaks on sizing columns. The first eluted peak contains a prominent 220-kDa protein. The second peak contains an apparently homogeneous 20-kDa polypeptide. A monoclonal antibody specific to rat brain STOP145 recognizes the 220-kDa protein, but not the 20-kDa species. The 220-kDa protein can be purified on a STOP antibody column and accounts for the bulk of stabilizing activity in the first peak. The 20-kDa protein does not bind to STOP antibody affinity columns. Sequence analysis of oligopeptide fragments of the 20-kDa protein shows 100% homology with bovine myelin basic protein (MBP). Anti-MBP antibodies recognize the 20-kDa, but not the 220-kDa species. We conclude that the 220-kDa protein is the bovine equivalent to rat brain STOP145 and that the 20-kDa species is MBP. Microtubule stabilization by MBP and STOP220 is abolished in the presence of Ca(2+)-calmodulin, and inhibition curves are similar for both proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adenylate cyclase activity in cyanobacteria: activation by Ca(2+)-calmodulin and a calmodulin-like activity

An adenylate cyclase activity was partially characterized in the cyanobacterium Anabaena sp. The enzyme activity is found in soluble cell fractions and shows an apparent molecular weight of about 183,400. This adenylate cyclase is activated by Ca2+ and bovine brain or spinach calmodulin and it is inhibited by EGTA and some phenothiazine derivatives. Furthermore, Anabaena sp. extracts contain a calmodulin-like activity which stimulates bovine brain cyclic AMP phosphodiesterase and the Anabaena adenylate cyclase. EGTA and phenothiazine derivatives block the cyanobacterial modulator effect.     

Molecular evolution and structural analysis of the Ca(2+) release-activated Ca(2+) channel subunit, Orai

Depletion of intracellular Ca(2+) stores evokes Ca(2+) entry across the plasma membrane by inducing Ca(2+) release-activated Ca(2+) (CRAC) currents in many cell types. Recently, Orai and STIM proteins were identified as the molecular identities of the CRAC channel subunit and the endoplasmic reticulum Ca(2+) sensor, respectively. Here, extensive database searching and phylogenetic analysis revealed several lineage-specific duplication events in the Orai protein family, which may account for the evolutionary origins of distinct functional properties among mammalian Orai proteins. Based on similarity to key structural domains and essential residues for channel functions in Orai proteins, database searching also identifies a putative primordial Orai sequence in hyperthermophilic archaeons. Furthermore, modern Orai appears to acquire new structural domains as early as Urochodata, before divergence into vertebrates. The evolutionary patterns of structural domains might be related to distinct functional properties of Drosophila and mammalian CRAC currents. Interestingly, Orai proteins display two conserved internal repeats located at transmembrane segments 1 and 3, both of which contain key amino acids essential for channel function. These findings demonstrate biochemical and physiological relevance of Orai proteins in light of different evolutionary origins and will provide novel insights into future structural and functional studies of Orai proteins.     

Phosphorylation of dystrophin and alpha-syntrophin by Ca(2+)-calmodulin dependent protein kinase II.

A Ca(2+)-calmodulin dependent protein kinase activity (DGC-PK) was previously shown to associate with skeletal muscle dystrophin glycoprotein complex (DGC) preparations, and phosphorylate dystrophin and a protein with the same electrophoretic mobility as alpha-syntrophin (R. Madhavan, H.W. Jarrett, Biochemistry 33 (1994) 5797-5804). Here, we show that DGC-PK and Ca(2+)-calmodulin dependent protein kinase II (CaM kinase II) phosphorylate a common site (RSDS(3616)) within the dystrophin C terminal domain that fits the consensus CaM kinase II phosphorylation motif (R/KXXS/T). Furthermore, both kinase activities phosphorylate exactly the same three fusion proteins (dystrophin fusions DysS7 and DysS9, and the syntrophin fusion) out of a panel of eight fusion proteins (representing nearly 100% of syntrophin and 80% of dystrophin protein sequences), demonstrating that DGC-PK and CaM kinase II have the same substrate specificity. Complementing these results, anti-CaM kinase II antibodies specifically stained purified DGC immobilized on nitrocellulose membranes. Renaturation of electrophoretically resolved DGC proteins revealed a single protein kinase band (M(r) approximately 60,000) that, like CaM kinase II, underwent Ca(2+)-calmodulin dependent autophosphorylation. Based on these observations, we conclude DGC-PK represents a dystrophin-/syntrophin-phosphorylating skeletal muscle isoform of CaM kinase II. We also show that phosphorylation of the dystrophin C terminal domain sequences inhibits their syntrophin binding in vitro, suggesting a regulatory role for phosphorylation.     

Inhibition of Acanthamoeba myosin I heavy chain kinase by Ca(2+)-calmodulin.

The actin-activated Mg(2+)-ATPase activity of Acanthamoeba myosins I depends on phosphorylation of their single heavy chains by myosin I heavy chain kinase. Kinase activity is enhanced > 50-fold by autophosphorylation at multiple sites. The rate of kinase autophosphorylation is increased approximately 20-fold by acidic phospholipids independent of the presence of Ca2+ and diglycerides. We show in this paper that Ca(2+)-calmodulin inhibits phospholipid-stimulated autophosphorylation of myosin I heavy chain kinase and hence also inhibits the catalytic activity of unphosphorylated kinase in the presence of phospholipid. Ca(2+)-calmodulin does not inhibit kinase activity in the absence of phospholipid. Micromolar Ca(2+)-calmodulin also inhibits binding of myosin I heavy chain kinase to phospholipid vesicles and purified plasma membranes. Proteolytic removal of a 7-kDa NH2-terminal segment from the 97-kDa kinase prevents binding of both calmodulin and phospholipid; therefore, we propose that they bind to the same or overlapping sites. These data provide a mechanism by which Ca2+ could inhibit the actin-activated Mg(2+)-ATPase activity of the myosin I isozymes in vivo and thus regulate myosin I-dependent motile activities.     

A structural model for the catalytic cycle of Ca(2+)-ATPase

Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule.     

Solution structure of Ca(2+)-calmodulin reveals flexible hand-like properties of its domains.

The solution structure of Ca(2+)-ligated calmodulin is determined from residual dipolar couplings measured in a liquid crystalline medium and from a large number of heteronuclear J couplings for defining side chains. Although the C-terminal domain solution structure is similar to the X-ray crystal structure, the EF hands of the N-terminal domain are considerably less open. The substantial differences in interhelical angles correspond to negligible changes in short interproton distances and, therefore, cannot be identified by comparison of NOEs and X-ray data. NOE analysis, however, excludes a two-state equilibrium in which the closed apo conformation is partially populated in the Ca(2+)-ligated state. The difference between the crystal and solution structures of Ca(2+)-calmodulin indicates considerable backbone plasticity within the domains of calmodulin, which is key to their ability to bind a wide range of targets. In contrast, the vast majority of side chains making up the target binding surface are locked into the same chi(1) rotameric states as in complexes with target peptide.     

Generation of specific Ca(2+) signals from Ca(2+) stores and endocytosis by differential coupling to messengers

It remains unclear how different intracellular stores could interact and be recruited by Ca(2+)-releasing messengers to generate agonist-specific Ca(2+) signatures. In addition, refilling of acidic stores such as lysosomes and secretory granules occurs through endocytosis, but this has never been investigated with regard to specific Ca(2+) signatures. In pancreatic acinar cells, acetylcholine (ACh), cholecystokinin (CCK), and the messengers cyclic ADP-ribose (cADPR), nicotinic acid adenine dinucleotide phosphate (NAADP), and inositol 1,4,5-trisphosphate (IP(3)) evoke repetitive local Ca(2+) spikes in the apical pole. Our work reveals that local Ca(2+) spikes evoked by different agonists all require interaction of acid Ca(2+) stores and the endoplasmic reticulum (ER), but in different proportions. CCK and ACh recruit Ca(2+) from lysosomes and from zymogen granules through different mechanisms; CCK uses NAADP and cADPR, respectively, and ACh uses Ca(2+) and IP(3), respectively. Here, we provide pharmacological evidence demonstrating that endocytosis is crucial for the generation of repetitive local Ca(2+) spikes evoked by the agonists and by NAADP and IP(3). We find that cADPR-evoked repetitive local Ca(2+) spikes are particularly dependent on the ER. We propose that multiple Ca(2+)-releasing messengers determine specific agonist-elicited Ca(2+) signatures by controlling the balance among different acidic Ca(2+) stores, endocytosis, and the ER.     

A mathematical model of cardiocyte Ca(2+) dynamics with a novel representation of sarcoplasmic reticular Ca(2+) control

Cardiac contraction and relaxation dynamics result from a set of simultaneously interacting Ca(2+) regulatory mechanisms. In this study, cardiocyte Ca(2+) dynamics were modeled using a set of six differential equations that were based on theories, equations, and parameters described in previous studies. Among the unique features of the model was the inclusion of bidirectional modulatory interplay between the sarcoplasmic reticular Ca(2+) release channel (SRRC) and calsequestrin (CSQ) in the SR lumen, where CSQ acted as a dynamic rather than simple Ca(2+) buffer, and acted as a Ca(2+) sensor in the SR lumen as well. The inclusion of this control mechanism was central in overcoming a number of assumptions that would otherwise have to be made about SRRC kinetics, SR Ca(2+) release rates, and SR Ca(2+) release termination when the SR lumen is assumed to act as a simple, buffered Ca(2+) sink. The model was sufficient to reproduce a graded Ca(2+)-induced Ca(2+) release (CICR) response, CICR with high gain, and a system with reasonable stability. As constructed, the model successfully replicated the results of several previously published experiments that dealt with the Ca(2+) dependence of the SRRC (, J. Gen. Physiol. 85:247-289), the refractoriness of the SRRC (, Am. J. Physiol. 270:C148-C159), the SR Ca(2+) load dependence of SR Ca(2+) release (, Am. J. Physiol. 268:C1313-C1329;, J. Biol. Chem. 267:20850-20856), SR Ca(2+) leak (, J. Physiol. (Lond.). 474:463-471;, Biophys. J. 68:2015-2022), SR Ca(2+) load regulation by leak and uptake (, J. Gen. Physiol. 111:491-504), the effect of Ca(2+) trigger duration on SR Ca(2+) release (, Am. J. Physiol. 258:C944-C954), the apparent relationship that exists between sarcoplasmic and sarcoplasmic reticular calcium concentrations (, Biophys. J. 73:1524-1531), and a variety of contraction frequency-dependent alterations in sarcoplasmic [Ca(2+)] dynamics that are normally observed in the laboratory, including rest potentiation, a negative frequency-[Ca(2+)] relationship, and extrasystolic potentiation. Furthermore, under the condition of a simulated Ca(2+) overload, an alternans-like state was produced. In summary, the current model of cardiocyte Ca(2+) dynamics provides an integrated theoretical framework of fundamental cellular Ca(2+) regulatory processes that is sufficient to predict a broad array of observable experimental outcomes.     

Postulated role of interdomain interaction within the ryanodine receptor in Ca(2+) channel regulation

Localized distribution of malignant hyperthermia (MH) and central core disease (CCD) mutations in N-terminal and central domains of the ryanodine receptor suggests that the interaction between these domains may be involved in Ca(2+) channel regulation. To test this hypothesis, we investigated the effects of a new synthetic domain peptide DP4 corresponding to the Leu(2442)-Pro(2477) region of the central domain. DP4 enhanced ryanodine binding and induced a rapid Ca(2+) release. The concentration for half-maximal activation by agonists was considerably reduced in the presence of DP4. These effects of DP4 are analogous to the functional modifications of the ryanodine receptor caused by MH/CCD mutations (viz. hyperactivation of the channel and hypersensitization of the channel to agonists). Replacement of Arg of DP4 with Cys, mimicking the in vivo Arg(2458)-to-Cys(2458) mutation, abolished the activating effects of DP4. An N-terminal domain peptide DP1 (El-Hayek, R., Saiki, Y., Yamamoto, T., and Ikemoto, N. (1999) J. Biol. Chem. 274, 33341-33347) shows similar activation/sensitization effects. The addition of both DP4 and DP1 produced mutual interference of their activating functions. We tentatively propose that contact between the two (N-terminal and central) domains closes the channel, whereas removal of the contact by these domain peptides or by MH/CCD mutations de-blocks the channel, resulting in hyperactivation/hyper-sensitization effects.     

p-Nitrophenylphosphatase activity catalyzed by plasma membrane (Ca(2+) + Mg(2+)ATPase: correlation with structural changes modulated by glycerol and Ca(2+)

The plasma membrane (Ca²⁺+Mg²⁺)ATPase hydrolyzes pseudo-substrates such as p-nitrophenylphosphate. Except when calmodulin is present, Ca²⁺ ions inhibit the p-nitrophenylphosphatase activity. In this report it is shown that, in the presence of glycerol, Ca²⁺ strongly stimulates phosphatase activity in a dose-dependent manner. The glycerol- and Ca²⁺-induced increase in activity is correlated with modifications in the spectral center of mass (average emission wavenumber) of the intrinsic fluorescence of the enzyme. It is concluded that the synergistic effect of glycerol and Ca²⁺ is related to opposite long-term hydration effects on the substrate binding domain and the Ca²⁺ binding domain.     

Intracellular Ca(2+) dynamics and the stability of ventricular tachycardia

Ventricular fibrillation (VF), the major cause of sudden cardiac death, is typically preceded by ventricular tachycardia (VT), but the mechanisms underlying the transition from VT to VF are poorly understood. Intracellular Ca(2+) overload occurs during rapid heart rates typical of VT and is also known to promote arrhythmias. We therefore studied the role of intracellular Ca(2+) dynamics in the transition from VT to VF, using a combined experimental and mathematical modeling approach. Our results show that 1) rapid pacing of rabbit ventricular myocytes at 35 degrees C led to increased intracellular Ca(2+) levels and complex patterns of action potential (AP) configuration and the intracellular Ca(2+) transients; 2) the complex patterns of the Ca(2+) transient arose directly from the dynamics of intracellular Ca(2+) cycling, and were not merely passive responses to beat-to-beat alterations in AP; 3) the complex Ca(2+) dynamics were simulated in a modified version of the Luo-Rudy (LR) ventricular action potential with improved intracellular Ca(2+) dynamics, and showed good agreement with the experimental findings in isolated myocytes; and 4) when incorporated into simulated two-dimensional cardiac tissue, this action potential model produced a form of spiral wave breakup from VT to a VF-like state in which intracellular Ca(2+) dynamics played a key role through its influence on Ca(2+)-sensitive membrane currents such as I(Ca), I(NaCa), and I(ns(Ca)). To the extent that spiral wave breakup is useful as a model for the transition from VT to VF, these findings suggest that intracellular Ca(2+) dynamics may play an important role in the destabilization of VT and its degeneration into VF.